Molecular characterization of human papillomavirus (HPV) associated oropharyngeal squamous cell carcinomas (OPSCC) is genetically complex but has provided some promising insight into individual genetic changes that contribute to improved cancer survival and tumor progression. However, current commercial methods for detecting HPV in tumors by histopathology have shown poor sensitivity and specificity when compared to the more labor intensive gold standard methods for detecting HPV RNA. Our research group has recently developed a novel 22 CpG loci panel whose methylation status distinguishes between OPSCC with and without HPV16 infections expressing the E6/E7 oncogenes. This has potential clinical relevance as detection of HPV16 E6/E7 RNA in tumors has been associated with significantly improved cancer survival in OPSCC. In this study, we propose to validate this host DNA methylation panel and test the novel epigenetic events that may regulate key effector proteins, including for four panel loci downstream of the transcriptional start site of CDKN2A(p16) that, despite being hypermethylated in tumor compared to normal tissues, are associated with increased expression of the cellular gene. In this study, we propose to test and validate this signature. We will: 1) test the hypothesis that changes in host DNA methylation status of the HPV panel have functional consequences in altering expression of their corresponding genes; 2) the hypothesis that HPV viral oncogenes E6 and E7 are sufficient to induce host DNA methylation and corresponding gene expression changes seen in the HPV panel, including the novel changes observed for CDKN2A(p16); and 3) test the clinical relevance of our HPV-driven methylation panel in clinical samples to determine which are most predictive of clinical outcome. Combined with our experiments establishing functional and clinical relevance, this study will lay the groundwork for future clinical studies to test clinical utility of an HPV-specific methylation panel.